Our understanding of protein localization and molecular interactions has been greatly enhanced through the use of fluorescent protein fusions. However, there are situations in which the large size (27 kD) of the fluorescent protein interferes with the physiological role of the protein under study (see, e.g., Andresen et al., Mol. Biol. Cell 2004, 15, 5616-5622, and Hoffmann et. al., J. Nat. Methods 2005, 2, 171-176). Furthermore, the spectral properties of fluorescent proteins are thus far restricted to the visible range (see, e.g., Shaner et al., Nat. Biotechnol. 2004, 22, 1567-1572) and their modest photostability has limited their use in many applications, such as single-molecule studies (see, e.g., Steinmeyer et al., J. Fluoresc. 2005, 15, 707-721).
Recently, a number of protein-based tags have been described that recruit small-molecule fluorophores through non-covalent or covalent interactions, both in vitro and in living cells (see, e.g., Los et al., Journal of Neurochemistry 2005, 94, 15-15, Miller et al., Nat Methods 2005, 2, 255-257, and Keppler et al., Nat. Biotechnol. 2003, 21, 86-89). Although these approaches have enabled the use of fluorophores with improved photophysical properties, these fusion proteins present a similarly large change in the size of the modified protein. On the other hand, strategies that utilize much smaller peptide tags—such as hexahistidine (H6) and tetracysteine (TC) motifs—have the potential to allow labeling with minimal perturbation of the protein itself (see, e.g., Lata et al., J. Am. Chem. Soc. 2006, 128, 2365-2372). Tetracysteine tags can be labeled intracellularly, but have thus far been practically limited to hydroxylated xanthene or phenoxazine dyes (e.g., FlAsH and ReAsH) (see, e.g., Griffin et al., Science 1998, 281, 269-272, Adams et al., J. Am. Chem. Soc. 2002, 124, 6063-6076, and Spagnuolo et al., J. Am. Chem. Soc. 2006, 128, 12040-12041).
The simultaneous structural requirements for both fluorescence and the rigid display of arsenic atoms have limited the range of fluorophores that can be targeted to tetracysteine tags. Although fluorescein and resorufin are compatible, their brightness, pH sensitivity and propensity to photobleach are sub-optimal. On the other hand, rhodamines are aminated xanthenes that are pH-insensitive, bright dyes with excellent photostability. However, bis-arsenical rhodamines have been reported to be non-fluorescent, even when bound to a tetracysteine tag (see, e.g., Adams et al., J. Am. Chem. Soc. 2002, 124, 6063-6076). Thus, the scope of compatible dyes can be both narrow and difficult to predict.